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Articles by M Li
Total Records ( 26 ) for M Li
  M Li , Y Chen , Z Liu , F Shen , X Bian and Y. Meng
 

Ribosome-inactivating proteins (RIPs) are a family of enzymes that depurinate rRNA and inhibit protein biosynthesis. Here we report the purification, apoptosis-inducing activity, and polyethylene glycol (PEG) modification of RIP from the bitter melon seeds. The protein has a homogenous N-terminal sequence of N-Asp-Val-Ser-Phe-Arg. Moreover, the RIP displayed strong apoptosis-inducing activity and suppressed cancer cell growth. This might be attributed to the activation of caspases-3. To make it available for in vivo application, the immunogenicity of RIP was reduced by chemical modification with 20 kDa (mPEG)2-Lys-NHS. The inhibition activity of both PEGylated and non-PEGylated RIP against cancer cells was much stronger than against normal cells, and the antigenicity of PEGylated RIP was reduced significantly. Our results suggested that the PEGylated RIP might be potentially developed as anti-cancer drug.

  T Fan , M Li , J Wang , L Yang and R. Cong
 

Phenoloxidase (PO) from ink sacs of Octopus ocellatus was purified by gel-filtration and ion-exchange chromatography, and characterized in terms of its biochemical and enzymatic properties by using L-dihydroxyphenylalanine (L-DOPA) as the specific substrate. It was found that prophenoloxidase from O. ocellatus was isolated as a heterodimeric protein of 153.8 kDa, and two subunits of 75.6 and 73.0 kDa were often detected in preparations after SDS activation. The PO-like activity showed optimal pH of 7.0, optimal temperature of 40°C, and an apparent Km value of 3.1 mM on L-DOPA, and 6.3 mM on catechol, respectively. The PO-like activity was extremely sensitive to 1-phenyl-2-thiourea and sodium sulfite, and very sensitive to ascorbic acid, thiourea, citric acid, and benzoic acid. Together with its specific enzyme activity on catechol and L-DOPA, it can be concluded that the Octopus PO is most probably a typical o-diphenoloxidase. The PO-like activity was also strongly inhibited by Cu2+, Zn2+, ethylenediaminetetraacetic acid and diethyldithiocarbamate (DETC), and the DETC-inhibited PO-like activity could be perfectly restored by Cu2+. These results indicated that Octopus PO is most probably a copper-containing metalloenzyme. All these results implied that the PO from O. ocellatus has the properties of a catechol-type copper-containing o-diphenoloxidase which functions not only as a catalytic enzyme in melanin production in ink sacs but also as a humoral factor in host defense via melaninization as in other crustaceans.

  P Tantiwong , K Shanmugasundaram , A Monroy , S Ghosh , M Li , R. A DeFronzo , E Cersosimo , A Sriwijitkamol , S Mohan and N. Musi
 

NF-B is a transcription factor that controls the gene expression of several proinflammatory proteins. Cell culture and animal studies have implicated increased NF-B activity in the pathogenesis of insulin resistance and muscle atrophy. However, it is unclear whether insulin-resistant human subjects have abnormal NF-B activity in muscle. The effect that exercise has on NF-B activity/signaling also is not clear. We measured NF-B DNA-binding activity and the mRNA level of putative NF-B-regulated myokines interleukin (IL)-6 and monocyte chemotactic protein-1 (MCP-1) in muscle samples from T2DM, obese, and lean subjects immediately before, during (40 min), and after (210 min) a bout of moderate-intensity cycle exercise. At baseline, NF-B activity was elevated 2.1- and 2.7-fold in obese nondiabetic and T2DM subjects, respectively. NF-B activity was increased significantly at 210 min following exercise in lean (1.9-fold) and obese (2.6-fold) subjects, but NF-B activity did not change in T2DM. Exercise increased MCP-1 mRNA levels significantly in the three groups, whereas IL-6 gene expression increased significantly only in lean and obese subjects. MCP-1 and IL-6 gene expression peaked at the 40-min exercise time point. We conclude that insulin-resistant subjects have increased basal NF-B activity in muscle. Acute exercise stimulates NF-B in muscle from nondiabetic subjects. In T2DM subjects, exercise had no effect on NF-B activity, which could be explained by the already elevated NF-B activity at baseline. Exercise-induced MCP-1 and IL-6 gene expression precedes increases in NF-B activity, suggesting that other factors promote gene expression of these cytokines during exercise.

  S Grealish , M. E Jonsson , M Li , D Kirik , A Bjorklund and L. H. Thompson
 

Grafts of foetal ventral mesencephalon, used in cell replacement therapy for Parkinson’s disease, are known to contain a mix of dopamine neuronal subtypes including the A9 neurons of the substantia nigra and the A10 neurons of the ventral tegmental area. However, the relative importance of these subtypes for functional repair of the brain affected by Parkinson’s disease has not been studied thoroughly. Here, we report results from a series of grafting experiments where the anatomical and functional properties of grafts either selectively lacking in A9 neurons, or with a typical A9/A10 composition were compared. The results show that the A9 component of intrastriatal grafts is of critical importance for recovery in tests on motor performance, in a rodent model of Parkinson’s disease. Analysis at the histological level indicates that this is likely to be due to the unique ability of A9 neurons to innervate and functionally activate their target structure, the dorsolateral region of the host striatum. The findings highlight dopamine neuronal subtype composition as a potentially important parameter to monitor in order to understand the variable nature of functional outcome better in transplantation studies. Furthermore, the results have interesting implications for current efforts in this field to generate well-characterized and standardized preparations of transplantable dopamine neuronal progenitors from stem cells.

  G Agiostratidou , M Li , K Suyama , I Badano , R Keren , S Chung , A Anzovino , J Hulit , B Qian , B Bouzahzah , E Eugenin , O Loudig , G. R Phillips , J Locker and R. B. Hazan
 

The mammary epithelium is thought to be stabilized by cell-cell adhesion mediated mainly by E-cadherin (E-cad). Here, we show that another cadherin, retinal cadherin (R-cad), is critical for maintenance of the epithelial phenotype. R-cad is expressed in nontransformed mammary epithelium but absent from tumorigenic cell lines. In vivo, R-cad was prominently expressed in the epithelium of both ducts and lobules. In human breast cancer, R-cad was down-regulated with tumor progression, with high expression in ductal carcinoma in situ and reduced expression in invasive duct carcinomas. By comparison, E-cad expression persisted in invasive breast tumors and cell lines where R-cad was lost. Consistent with these findings, R-cad knockdown in normal mammary epithelium stimulated invasiveness and disrupted formation of acini despite continued E-cad expression. Conversely, R-cad overexpression in aggressive cell lines induced glandular morphogenesis and inhibited invasiveness, tumor formation, and lung colonization. R-cad also suppressed the matrix metalloproteinase 1 (MMP1), MMP2, and cyclooxygenase 2 gene expression associated with pulmonary metastasis. The data suggest that R-cad is an adhesion molecule of the mammary epithelium, which acts as a critical regulator of the normal phenotype. As a result, R-cad loss contributes to epithelial suppression and metastatic progression. [Cancer Res 2009;69(12):5030–8]

  M Li , H Edamatsu , R Kitazawa , S Kitazawa and T. Kataoka
 

ApcMin/+ mice, carrying an inactivated allele of the adenomatous polyposis coli gene, are widely used as an animal model for human colorectal tumorigenesis, where tumor environment, such as inflammation, is known to play a critical role in tumor progression. We previously demonstrated that phospholipase C (PLC), an effector of Ras and Rap small GTPases, plays a crucial role in two-stage skin chemical carcinogenesis using 12-O-tetradecanoyl-phorbor-13-acetate (TPA) as a promoter through augmentation of TPA-induced inflammation. Here, we show that ApcMin/+ mice lacking PLC (PLC–/–) exhibit marked resistance to spontaneous intestinal tumorigenesis compared with those with the PLC+/+ background. Time course of the development of tumors, which are histopathologically classified into low- and high-grade adenomas with increasing dysplasia and size, and adenocarcinomas indicates that not only the low-grade adenoma formation but also the progression to high-grade adenoma are suppressed in PLC–/–;ApcMin/+ mice. Low-grade adenomas of PLC–/–;ApcMin/+ mice exhibit accelerated apoptosis and reduced cellular proliferation. They also show marked attenuation of tumor angiogenesis and reduction in expression of vascular endothelial growth factor. In contrast, high-grade adenomas of PLC–/–;ApcMin/+ mice exhibit marked attenuation of tumor-associated inflammation without significant differences in apoptosis and proliferation. These results suggest that PLC plays crucial roles in intestinal tumorigenesis through two distinct mechanisms, augmentation of angiogenesis and inflammation, depending on the tumor stage.

  J Wang , Q Gu , M Li , W Zhang , M Yang , B Zou , S Chan , L Qiao , B Jiang , S Tu , J Ma , I. F Hung , H. Y Lan and B. C.Y. Wong
 

Background and aims: X-linked inhibitor of apoptosis-associated factor 1 (XAF1) was first recognized as an antagonist of X-linked inhibitor of apoptosis in suppressing caspase 3 activity. It has lower expression in cancer cells than normal tissue. Overexpression of XAF1 can inhibit cancer cell growth and sensitize tumor necrosis factor-related apoptosis-inducing ligand- or etoposide-induced apoptosis. The aim of this study is to elucidate the mechanism of XAF1 in regulating cell growth. Methods: Stable transfectants of gastrointestinal (GI) cancer cell lines AGS and SW1116 expressing XAF1 and vector control were generated. Cell growth, apoptosis, mitotic status and cell cycle distribution were assessed. The interaction between XAF1 and G2/M checkpoint proteins was evaluated by immunoblotting, kinase assay and co-immunoprecipitation assay. Mitotic catastrophe was identified by occurrence of aberrant nuclei and centrosomal amplification. Results: Our results showed that overexpression of XAF1 suppressed serum-dependent cancer cell growth, induced mitotic catastrophe and G2/M cell cycle arrest. Interestingly, XAF1 was predominantly expressed in G2/M phase after cell cycle synchronization. XAF1 interacted with and activated checkpoint kinase 1 (Chk1), inactivated Cdc25C and lead to inactivation of Cdc2–cyclin B complex. Suppression of Chk1 abrogated XAF1-induced G2/M arrest. Conclusions: Our findings implicate XAF1 as a novel cell cycle modulator that is recruited in G2/M phase and thus unravel a novel function pathway of XAF1, suggesting the potential role of XAF1 as the target for the management of GI cancers.

  T. P Cappola , M Li , J He , B Ky , J Gilmore , L Qu , B Keating , M Reilly , C. E Kim , J Glessner , E Frackelton , H Hakonarson , F Syed , A Hindes , S. J Matkovich , S Cresci and G. W. Dorn
 

Background— Heart failure results from abnormalities in multiple biological processes that contribute to cardiac dysfunction. We tested the hypothesis that inherited variation in genes of known importance to cardiovascular biology would thus contribute to heart failure risk.

Methods and Results— We used the ITMAT/Broad/CARe cardiovascular single-nucleotide polymorphism array to screen referral populations of patients with advanced heart failure for variants in 2000 genes of predicted importance to cardiovascular biology. Our design was a 2-stage case-control study. In stage 1, genotypes in Caucasian patients with heart failure (n=1590; ejection fraction, 32±16%) were compared with those in unaffected controls (n=577; ejection fraction, 67±8%) who were recruited from the same referral centers. Associations were tested for independent replication in stage 2 (308 cases and 2314 controls). Two intronic single-nucleotide polymorphisms showed replicated associations with all-cause heart failure as follows: rs1739843 in HSPB7 (combined P=3.09x10–6) and rs6787362 in FRMD4B (P=6.09x10–6). For both single-nucleotide polymorphisms, the minor allele was protective. In subgroup analyses, rs1739843 associated with both ischemic and nonischemic heart failure, whereas rs6787362 associated principally with ischemic heart failure. Linkage disequilibrium surrounding rs1739843 suggested that the causal variant resides in a region containing HSPB7 and a neighboring gene, CLCNKA, whereas the causal variant near rs6787362 is probably within FRMD4B. Allele frequencies for these single-nucleotide polymorphisms were substantially different in African Americans (635 cases and 714 controls) and showed no association with heart failure in this population.

Conclusions— Our findings identify regions containing HSPB7 and FRMD4B as novel susceptibility loci for advanced heart failure. More broadly, in an era of genome-wide association studies, we demonstrate how knowledge of candidate genes can be leveraged as a complementary strategy to discern the genetics of complex disorders.

  M Preuss , I. R Konig , J. R Thompson , J Erdmann , D Absher , T. L Assimes , S Blankenberg , E Boerwinkle , L Chen , L. A Cupples , A. S Hall , E Halperin , C Hengstenberg , H Holm , R Laaksonen , M Li , W Marz , R McPherson , K Musunuru , C. P Nelson , M Susan Burnett , S. E Epstein , C. J O'Donnell , T Quertermous , D. J Rader , R Roberts , A Schillert , K Stefansson , A. F. R Stewart , G Thorleifsson , B. F Voight , G. A Wells , A Ziegler , S Kathiresan , M. P Reilly , N. J Samani , H Schunkert and on behalf of the CARDIoGRAM Consortium
  Background—

Recent genome-wide association studies (GWAS) of myocardial infarction (MI) and other forms of coronary artery disease (CAD) have led to the discovery of at least 13 genetic loci. In addition to the effect size, power to detect associations is largely driven by sample size. Therefore, to maximize the chance of finding novel susceptibility loci for CAD and MI, the Coronary ARtery DIsease Genome-wide Replication And Meta-analysis (CARDIoGRAM) consortium was formed.

Methods and Results—

CARDIoGRAM combines data from all published and several unpublished GWAS in individuals with European ancestry; includes >22 000 cases with CAD, MI, or both and >60 000 controls; and unifies samples from the Atherosclerotic Disease VAscular functioN and genetiC Epidemiology study, CADomics, Cohorts for Heart and Aging Research in Genomic Epidemiology, deCODE, the German Myocardial Infarction Family Studies I, II, and III, Ludwigshafen Risk and Cardiovascular Heath Study/AtheroRemo, MedStar, Myocardial Infarction Genetics Consortium, Ottawa Heart Genomics Study, PennCath, and the Wellcome Trust Case Control Consortium. Genotyping was carried out on Affymetrix or Illumina platforms followed by imputation of genotypes in most studies. On average, 2.2 million single nucleotide polymorphisms were generated per study. The results from each study are combined using meta-analysis. As proof of principle, we meta-analyzed risk variants at 9p21 and found that rs1333049 confers a 29% increase in risk for MI per copy (P=2x10–20).

Conclusion—

CARDIoGRAM is poised to contribute to our understanding of the role of common genetic variation on risk for CAD and MI.

  Q Sun , M Hang , W Xu , W Mao , X Hang , M Li and J. Zhang
  Objective

This Phase II study was conducted to evaluate the effects of irinotecan plus capecitabine in patients with advanced gastric cancer (AGC) who had received a first-line therapy of 5-fluorouracil/platinum regimen.

Methods

Patients received capecitabine 1000 mg/m2 b.i.d. on days 1–14 followed by a 7-day rest period, and irinotecan 100 mg/m2 was administered through a 90 min intravenous infusion on days 1 and 8, based on a 3-week cycle.

Results

Forty-six (95.8%) of the 48 patients were assessable for response. Thirteen cases of partial response were confirmed, response rate of 27.1% (95% CI, 14.5–39.7%). The median follow-up period was 25.2 months. The median time to progression and overall survival for all patients were 4.1 months (95% CI, 3.4–4.8 months) and 7.6 months (95% CI, 5.1–10.1 months). Grade 3 diarrhea and hand-foot syndrome occurred in eight (17.4%) and two (4.3%) patients, respectively. The most common Grade 3/4 hematological adverse event was neutropenia in four (8.7%) patients. There were no treatment-related deaths during this study.

Conclusion

Irinotecan plus capecitabine was a relatively active and tolerable regimen as a second-line chemotherapy for AGC. Further investigation of this regimen is warranted, including the addition of new biological agents such as bevacizumab or cetuximab to improve the salvage regimen.

  Q Lin , M Li , Z. Y Shen , L. W Xiong , X. F Pan , J. F Gen , G. L Bao , H. F Sha , J. X Feng , C. Y Ji and M. Chen
  Objective

The purpose of this study was to evaluate the value of vascular endothelial growth factor-A and E-cadherin expression as well as other confirmed prognostic factors in predicting the clinical outcome after definitive surgery of pathologic stage I non-small cell lung cancer.

Methods

One hundred and eighty-five consecutive and non-selected patients who underwent definitive surgery for stage I non-small cell lung cancer in our institute were included in this study. Formalin-fixed paraffin-embedded specimens were stained for vascular endothelial growth factor-A and E-cadherin and the correlation between the staining, its clinicopathological parameters and its prognostic power were analyzed statistically.

Results

Of the 185 patients studied, 92 cases (49.7%) were strongly positive for vascular endothelial growth factor-A. Vascular endothelial growth factor-A expression was only related to visceral pleural involvement (P < 0.001). A total of 95 carcinomas (51.4%) were E-cadherin-negative tumors. E-cadherin expression correlated with histology (P < 0.001), tumor size (P = 0.001) and visceral pleural involvement (P < 0.001). In univariate analysis by log-rank test, gender, tumor size, lymphovascular invasion, visceral pleural involvement, vascular endothelial growth factor-A expression and E-cadherin expression were significant prognostic factors (P = 0.003, 0.042, 0.026, 0.035, 0.008 and 0.006, respectively). In multivariate analysis, gender, vascular endothelial growth factor-A and E-cadherin expression maintained its independent prognostic influence on overall survival (P = 0.013, <0.001 and 0.036, respectively).

Conclusions

Expression of vascular endothelial growth factor-A is related to visceral pleural involvement, and E-cadherin expression correlates with histology, tumor size and visceral pleural involvement. Multivariate analysis confirmed gender, vascular endothelial growth factor-A and E-cadherin expression were significant predictive factors for overall survival in completely resected pathologic stage I non-small cell lung cancer.

  G Di Leva , P Gasparini , C Piovan , A Ngankeu , M Garofalo , C Taccioli , M. V Iorio , M Li , S Volinia , H Alder , T Nakamura , G Nuovo , Y Liu , K. P Nephew and C. M. Croce
  Background

Several lines of evidence have suggested that estrogen receptor (ER)–negative breast tumors, which are highly aggressive and nonresponsive to hormonal therapy, arise from ER-positive precursors through different molecular pathways. Because microRNAs (miRNAs) modulate gene expression, we hypothesized that they may have a role in ER-negative tumor formation.

Methods

Gene expression profiles were used to highlight the global changes induced by miRNA modulation of ER protein. miRNA transfection and luciferase assays enabled us to identify new targets of miRNA 206 (miR-206) and miRNA cluster 221-222 (miR-221-222). Northern blot, luciferase assays, estradiol treatment, and chromatin immunoprecipitation were performed to identify the miR-221-222 transcription unit and the mechanism implicated in its regulation.

Results

Different global changes in gene expression were induced by overexpression of miR-221-222 and miR-206 in ER-positive cells. miR-221 and -222 increased proliferation of ER-positive cells, whereas miR-206 had an inhibitory effect (mean absorbance units [AU]: miR-206: 500 AU, 95% confidence interval [CI]) = 480 to 520; miR-221: 850 AU, 95% CI = 810 to 873; miR-222: 879 AU, 95% CI = 850 to 893; P < .05). We identified hepatocyte growth factor receptor and forkhead box O3 as new targets of miR-206 and miR-221-222, respectively. We demonstrated that ER negatively modulates miR-221 and -222 through the recruitment of transcriptional corepressor partners: nuclear receptor corepressor and silencing mediator of retinoic acid and thyroid hormone receptor.

Conclusions

These findings suggest that the negative regulatory loop involving miR-221-222 and ER may confer proliferative advantage and migratory activity to breast cancer cells and promote the transition from ER-positive to ER-negative tumors.

  S Ubaidus , M Li , S Sultana , P. H. L de Freitas , K Oda , T Maeda , R Takagi and N. Amizuka
 

This study aimed to evaluate whether the immunolocalization of fibroblast growth factor (FGF) 23 and dentin matrix protein 1 (DMP1) is associated with the spatial regularity of the osteocyte lacunar canalicular system(s) (OLCS). Femora of 12-weeks-old male ICR mice were fixed with 4% paraformaldehyde, decalcified with a 10% EDTA solution and then embedded in paraffin. We have devised a triple staining procedure that combines silver impregnation, alkaline phosphatase (ALPase) immunohistochemistry and tartrate-resistant acid phosphatase (TRAPase) enzyme histochemistry on a single paraffin section. This procedure permitted the visualization of ALPase-positive plump osteoblasts and several TRAPase-positive osteoclasts on those bone matrices featuring irregularly arranged OLCS, and of ALPase-positive bone lining cells on the bone matrix displaying the well-arranged OLCS. As observations proceeded from the metaphysis toward the diaphysis, the endosteal cortical bone displayed narrower bands of calcein labeling, accompanied by increased regularity of the OLCS. This implies that the speed of bone deposition during bone remodeling would affect the regularity of the OLCS. While DMP1 was evenly localized in all regions of the cortical bones, FGF23 was more abundantly localized in osteocytes of cortical bones with regularly arranged OLCS. In cortical bones, the endosteal area featuring regular OLCS exhibited more intense FGF23 immunoreaction when compared to the periosteal region, which tended to display irregular OLCS. In summary, FGF23 appears to be synthesized principally by osteocytes in the regularly distributed OLCS that have been established after bone remodeling.

  M Li , Y Seki , P. H. L Freitas , M Nagata , T Kojima , S Sultana , S Ubaidus , T Maeda , J Shimomura , J. E Henderson , M Tamura , K Oda , Z Liu , Y Guo , R Suzuki , T Yamamoto , R Takagi and N. Amizuka
 

The signaling axis comprising the parathyroid hormone (PTH)-related peptide (PTHrP), the PTH/PTHrP receptor and the fibroblast growth factor receptor 3 (FGFR3) plays a central role in chondrocyte proliferation. The Indian hedgehog (IHH) gene is normally expressed in early hypertrophic chondrocytes, and its negative feedback loop was shown to regulate PTH/PTHrP receptor signaling. In this study, we examined the regulation of PTH/PTHrP receptor gene expression in a FGFR3-transfected chondrocytic cell line, CFK2. Expression of IHH could not be verified on these cells, with consequent absence of hypertrophic differentiation. Also, expression of the PTH/PTHrP receptor (75% reduction of total mRNA) and the PTHrP (50% reduction) genes was reduced in CFK2 cells transfected with FGFR3 cDNA. Interestingly, we verified significant reduction in cell growth and increased apoptosis in the transfected cells. STAT1 was detected in the nuclei of the CFK2 cells transfected with FGFR3 cDNA, indicating predominance of the JAK/STAT signaling pathway. The reduction in PTH/PTHrP receptor gene in CFK2 cells overexpressing FGFR3 was partially blocked by treatment with an inhibitor of JAK3 (WHI-P131), but not with an inhibitor of MAPK (SB203580) or JAK2 (AG490). Altogether, these findings suggest that FGFR3 down-regulates PTH/PTHrP receptor gene expression via the JAK/STAT signaling in chondrocytic cells.

  K Narimatsu , M Li , P. H. L de Freitas , S Sultana , S Ubaidus , T Kojima , L Zhucheng , G Ying , R Suzuki , T Yamamoto , K Oda and N. Amizuka
 

Preosteoblasts are currently defined as the precursors of mature osteoblasts. These cells are morphologically diverse and may represent a continuum during osteoblast differentiation. We have attempted to categorize the different preosteoblastic phenotypes in vivo by examining bone cells expressing the runt-related transcription factor 2, alkaline phosphatase and BrdU incorporation – histological traits of a preosteoblast – under transmission electron microscopy (TEM). TEM observations demonstrated, at least, in part two preosteoblastic subtypes: (i) a cell rich in cisterns of rough endoplasmic reticulum (rER) with vesicles and vacuoles and (ii) a subtype featuring extended cytoplasmic processes that connect with distant cells, with a small amount of scattered cisterns of rER and with many vesicles and vacuoles. ER-rich cells, whose cellular machinery is similar to that of an osteoblast, were often seen adjacent to mature osteoblasts, and therefore, may be ready for terminal differentiation. In contrast, ER-poor and vesicle-rich cells extended their cytoplasmic processes to mature osteoblasts and, frequently, to bone-resorbing osteoclasts. The abundant vesicles and vacuoles identified in this cell type indicate that this cell is involved in vesicular transport rather than matrix synthesis activity. In summary, our study verified the morphological diversity and the ultrastructural properties of osteoblastic cells in vivo.

  Z Xiao , G Li , Y Chen , M Li , F Peng , C Li , F Li , Y Yu , Y Ouyang and Z. Chen
 

Formalin-fixed, paraffin-embedded (FFPE) tissue specimens represent a potentially valuable resource for protein biomarker investigations. In this study, proteins were extracted by a heat-induced antigen retrieval technique combined with a retrieval solution containing 2% SDS from FFPE tissues of normal nasopharyngeal epithelial tissues (NNET) and three histological types of nasopharyngeal carcinoma (NPC) with diverse differentiation degrees. Then two-dimensional liquid chromatography-tandem mass spectrometry coupled with isobaric tags for relative and absolute quantification (iTRAQ) labeling was employed to quantitatively identify the differentially expressed proteins among the types of NPC FFPE tissues. Our study resulted in the identification of 730 unique proteins, the distributions of subcellular localizations and molecular functions of which were similar to those of the proteomic database of human NPC and NNET that we had set up based on the frozen tissues. Additionally, the relative expression levels of cathepsin D, keratin8, SFN, and stathmin1 identified and quantified in this report were consistent with the immunohistochemistry results acquired in our previous study. In conclusion, we have developed an effective approach to identifying protein changes in FFPE NPC tissues utilizing iTRAQ technology in conjunction with an economical and easily accessible sample preparation method. (J Histochem Cytochem 58:517–527, 2010)

  S. J Peterson , D. H Kim , M Li , V Positano , L Vanella , L. F Rodella , F Piccolomini , N Puri , A Gastaldelli , C Kusmic , A L'Abbate and N. G. Abraham
 

We examined mechanisms by which L-4F reduces obesity and diabetes in obese (ob) diabetic mice. We hypothesized that L-4F reduces adiposity via increased pAMPK, pAKT, HO-1, and increased insulin receptor phosphorylation in ob mice. Obese and lean mice were divided into five groups: lean, lean-L-4F-treated, ob, ob-L-4F-treated, and ob-L-4F-LY294002. Food intake, insulin, glucose adipocyte stem cells, pAMPK, pAKT, CB1, and insulin receptor phosphorylation were determined. Subcutaneous (SAT) and visceral adipose tissue (VAT) were determined by MRI and hepatic lipid content by magnetic resonance spectroscopy. SAT and VAT volumes decreased in ob-L-4F-treated animals compared with control. L-4F treatment decreased hepatic lipid content and increased the numbers of small adipocytes (P < 0.05) and phosphorylation of insulin receptors. L-4F decreased CB1 in SAT and VAT and increased pAKT and pAMPK in endothelium. L-4F-mediated improvement in endothelium was prevented by LY294002. Inhibition of pAKT and pAMPK by LY294002 was associated with an increase in glucose levels. Upregulation of HO-1 by L-4F produced adipose remodeling and increased the number of small differentiated adipocytes. The anti-obesity effects of L-4F are manifested by a decrease in visceral fat content with reciprocal increases in adiponectin, pAMPK, pAKT, and phosphorylation of insulin receptors with improved insulin sensitivity.

  H Zhang , M Li , X Zheng , Y Sun , Z Wen and X. Zhao
 

In normal endometrium, stromal factors regulate the growth of epithelial cells. However, epithelial cells in endometriotic lesions display increased proliferation and decreased apoptosis. This work tested the hypothesis that in endometriosis stromal cells lose the ability to regulate survival signaling and cell growth in epithelial cells. Primary normal, endometriotic eutopic and ectopic epithelial cells were cultured in the presence of medium conditioned by normal, eutopic and ectopic endometriotic endometrial stromal cells. Endometriotic epithelial cells showed higher Survivin expression than normal epithelial cells. Conditioned medium (CM) from normal or eutopic endometriotic stromal cells significantly inhibited the Survivin expression and AKt phosphorylation in normal or eutopic endometriotic epithelial cells. However, CM from ectopic endometriotic stromal cells did not have an inhibitory effect on normal or ectopic endometriotic epithelial cells. Inhibition of AKt phosphorylation and Survivin expression in normal or eutopic endometriotic epithelial cells in the presence of stromal factors from normal or eutopic endometriotic stromal cells was enhanced by progesterone, whereas progesterone had little effect in the presence of stromal factors from ectopic endometriotic stromal cells. The inability of ectopic endometriotic stromal cells to regulated PI3K/AKt/Survivin signaling and mediate the progesterone response in endometriotic epithelial cells may facilitate epithelial cell proliferation in endometriosis and promote the survival of endometriotic lesions.

  M Li , Y Sun , J. M Simard , J. Y Wang and T. C. Chai
 

Overactive bladder syndrome (OAB) is an idiopathic condition characterized by urinary urgency and urge incontinence. Detrusor overactivity has been traditionally described as the physiologic mechanism for OAB. However, the bladder urothelium (BU) may also be involved in the pathophysiology. This study measured polyamine signaling and its downstream effects on membrane conductivity in bladder urothelial cells (BUC) obtained from asymptomatic and OAB subjects. Immunohistofluorescence was used to measure ornithine decarboxylase (ODC) expression in BU. BUC, cultured from BU biopsies, were used for electrophysiologic studies. dl--Difluoromethylornithine (DFMO), spermine, or spermidine was used to modulate polyamine signaling in BUC. Results showed ODC overexpression in OAB BU. In OAB BUC, whole cell and cell-attached configuration showed significantly decreased currents. Using inside-out patches, outward currents increased significantly, suggesting a cytoplasmic source of the outward current block in OAB BUC. In control BUC, outward currents were mediated by the large-conductance calcium-activated potassium (BK) channel due to calcium dose-dependence and block by iberiotoxin. Spermidine and spermine blocked the outward current in normal BUC in dose-dependent fashion. Conversely, DFMO significantly increased (P < 0.01) outward currents in OAB BUC both in cell-attached and in whole cell configuration. The outward currents in DFMO-treated-OAB BUC could be significantly reduced (P < 0.05) by adding back spermidine and spermine. These data suggest that polyamine signaling is upregulated in OAB urothelium and OAB BUC. Furthermore, polyamines in BUC block the BK channel. Targeting of bladder urothelial polyamine signaling may represent a novel approach for OAB treatment based on pathophysiologic mechanisms.

  D Sheng , J Jao , M Li , P Xu and J. Zhang
 

Apart from inhibiting RecA activity through protein–protein interactions, Deinococcus radiodurans RecX inhibits the expression of RecA and two other anti-oxidant proteins. To identify the repertoire of proteins regulated by RecX, comparative proteomic studies were undertaken on a wild-type strain (R1) and recX null mutant (RecX). Two-dimensional electrophoresis followed by MALDI-TOF identification revealed 35 differentially expressed proteins, including 12 up-regulated and 23 down-regulated proteins in the mutant. The 12 up-regulated proteins are DNA repair proteins, stress response proteins, and metabolism-related proteins. Most of these have been previously characterized as ionizing radiation-induced proteins. The 23 down-regulated proteins are mainly involved in cellular metabolism, and some of these are key enzymes in the metabolic pathway. Thus, RecX is suggested to be involved in the switch between DNA damage response and normal metabolism in D. radiodurans.

  M Li , X Fang , Z Wei , J. P York and P. Zhang
 

Genomic instability is a hallmark of human cancers. Spindle assembly checkpoint (SAC) is a critical cellular mechanism that prevents chromosome missegregation and therefore aneuploidy by blocking premature separation of sister chromatids. Thus, SAC, much like the DNA damage checkpoint, is essential for genome stability. In this study, we report the generation and analysis of mice carrying a Cdc20 allele in which three residues critical for the interaction with Mad2 were mutated to alanine. The mutant Cdc20 protein (AAA-Cdc20) is no longer inhibited by Mad2 in response to SAC activation, leading to the dysfunction of SAC and aneuploidy. The dysfunction could not be rescued by the additional expression of another Cdc20 inhibitor, BubR1. Furthermore, we found that Cdc20AAA/AAA mice died at late gestation, but Cdc20+/AAA mice were viable. Importantly, Cdc20+/AAA mice developed spontaneous tumors at highly accelerated rates, indicating that the SAC-mediated inhibition of Cdc20 is an important tumor-suppressing mechanism.

  L Chorro , A Sarde , M Li , K. J Woollard , P Chambon , B Malissen , A Kissenpfennig , J. B Barbaroux , R Groves and F. Geissmann
 

Most tissues develop from stem cells and precursors that undergo differentiation as their proliferative potential decreases. Mature differentiated cells rarely proliferate and are replaced at the end of their life by new cells derived from precursors. Langerhans cells (LCs) of the epidermis, although of myeloid origin, were shown to renew in tissues independently from the bone marrow, suggesting the existence of a dermal or epidermal progenitor. We investigated the mechanisms involved in LC development and homeostasis. We observed that a single wave of LC precursors was recruited in the epidermis of mice around embryonic day 18 and acquired a dendritic morphology, major histocompatibility complex II, CD11c, and langerin expression immediately after birth. Langerin+ cells then undergo a massive burst of proliferation between postnatal day 2 (P2) and P7, expanding their numbers by 10–20-fold. After the first week of life, we observed low-level proliferation of langerin+ cells within the epidermis. However, in a mouse model of atopic dermatitis (AD), a keratinocyte signal triggered increased epidermal LC proliferation. Similar findings were observed in epidermis from human patients with AD. Therefore, proliferation of differentiated resident cells represents an alternative pathway for development in the newborn, homeostasis, and expansion in adults of selected myeloid cell populations such as LCs. This mechanism may be relevant in locations where leukocyte trafficking is limited.

  L Jones , G Wei , S Sevcikova , V Phan , S Jain , A Shieh , J. C. Y Wong , M Li , J Dubansky , M. L Maunakea , R Ochoa , G Zhu , T. R Tennant , K. M Shannon , S. W Lowe , M. M Le Beau and S. C. Kogan
 

Gain of chromosome 8 is the most common chromosomal gain in human acute myeloid leukemia (AML). It has been hypothesized that gain of the MYC protooncogene is of central importance in trisomy 8, but the experimental data to support this are limited and controversial. In a mouse model of promyelocytic leukemia in which the MRP8 promoter drives expression of the PML-RARA fusion gene in myeloid cells, a Myc allele is gained in approximately two-thirds of cases as a result of trisomy for mouse chromosome 15. We used this model to test the idea that MYC underlies acquisition of trisomy in AML. We used a retroviral vector to drive expression of wild-type, hypermorphic, or hypomorphic MYC in bone marrow that expressed the PML-RARA transgene. MYC retroviruses cooperated in myeloid leukemogenesis and suppressed gain of chromosome 15. When the PML-RARA transgene was expressed in a Myc haploinsufficient background, we observed selection for increased copies of the wild-type Myc allele concomitant with leukemic transformation. In addition, we found that human myeloid leukemias with trisomy 8 have increased MYC. These data show that gain of MYC can contribute to the pathogenic effect of the most common trisomy of human AML.

  M. F Tsai , M Li and T. C. Hwang
 

Cystic fibrosis transmembrane conductance regulator (CFTR), a member of the adenosine triphosphate (ATP) binding cassette (ABC) superfamily, is an ATP-gated chloride channel. Like other ABC proteins, CFTR encompasses two nucleotide binding domains (NBDs), NBD1 and NBD2, each accommodating an ATP binding site. It is generally accepted that CFTR’s opening–closing cycles, each completed within 1 s, are driven by rapid ATP binding and hydrolysis events in NBD2. Here, by recording CFTR currents in real time with a ligand exchange protocol, we demonstrated that during many of these gating cycles, NBD1 is constantly occupied by a stably bound ATP or 8-N3-ATP molecule for tens of seconds. We provided evidence that this tightly bound ATP or 8-N3-ATP also interacts with residues in the signature sequence of NBD2, a telltale sign for an event occurring at the NBD1–NBD2 interface. The open state of CFTR has been shown to represent a two-ATP–bound NBD dimer. Our results indicate that upon ATP hydrolysis in NBD2, the channel closes into a "partial NBD dimer" state where the NBD interface remains partially closed, preventing ATP dissociation from NBD1 but allowing the release of hydrolytic products and binding of the next ATP to occur in NBD2. Opening and closing of CFTR can then be coupled to the formation and "partial" separation of the NBD dimer. The tightly bound ATP molecule in NBD1 can occasionally dissociate from the partial dimer state, resulting in a nucleotide-free monomeric state of NBDs. Our data, together with other structural/functional studies of CFTR’s NBDs, suggest that this process is poorly reversible, implying that the channel in the partial dimer state or monomeric state enters the open state through different pathways. We therefore proposed a gating model for CFTR with two distinct cycles. The structural and functional significance of our results to other ABC proteins is discussed.

  Z Kopeikin , Y Sohma , M Li and T. C. Hwang
 

The effects of a thiazolidinone derivative, 3-[(3-trifluoromethyl)phenyl]-5-[(4-carboxyphenyl)methylene]-2-thioxo-4-thiazolidinone (or CFTRinh-172), on cystic fibrosis transmembrane conductance regulator (CFTR) gating were studied in excised inside-out membrane patches from Chinese hamster ovary cells transiently expressing wild-type and mutant CFTR. We found that the application of CFTRinh-172 results in an increase of the mean closed time and a decrease of the mean open time of the channel. A hyperbolic relationship between the closing rate and [CFTRinh-172] suggests that CFTRinh-172 does not act as a simple pore blocker. Interestingly, the potency of inhibition increases as the open time of the channel is increased with an IC50 in the low nanomolar range for CFTR channels locked in an open state for tens of seconds. Our studies also provide evidence that CFTRinh-172 can bind to both the open state and the closed state. However, at least one additional step, presumably reflecting inhibitor-induced conformational changes, is required to shut down the conductance after the binding of the inhibitor to the channel. Using the hydrolysis-deficient mutant E1371S as a tool as the closing rate of this mutant is dramatically decreased, we found that CFTRinh-172–dependent inhibition of CFTR channel gating, in two aspects, mimics the inactivation of voltage-dependent cation channels. First, similar to the recovery from inactivation in voltage-gated channels, once CFTR is inhibited by CFTRinh-172, reopening of the channel can be seen upon removal of the inhibitor in the absence of adenosine triphosphate (ATP). Second, ATP induced a biphasic current response on inhibitor-bound closed channels as if the ATP-opened channels "inactivate" despite a continuous presence of ATP. A simplified six-state kinetic scheme can well describe our data, at least qualitatively. Several possible structural mechanisms for the effects of CFTRinh-172 will be discussed.

 
 
 
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